The ultimate goal of islet transplantation is to achieve tolerance defined as long-tem engraftment without maintenance immunosuppression. Several studies suggest that certain extracellular vesicles (exosomes) play an essential role in the immune responses involved in both rejection and tolerance of allogeneic transplants. Recently, we reported that, after pancreatic islet transplantation in mice, many recipient cells, take up donor vesicles and present allogeneic MHC molecules on their surface (allo-MHC cross-dressing); a process leading to activation of alloreactive T cells in vivo. In addition, exosomes released by pancreatic insulin-secreting beta cells are known to promote inflammation and diabetes through transfer of auto-antigens and RNA. Altogether, this suggests that exosomes might play a key role in the rejection of islet allografts. Supporting this view, we have obtained preliminary evidence that in vivo blocking of donor exosome production with 2 agents, GW4869 and cambinol, inhibited allo-MHC cross-dressing and prolonged survival of heart allografts in mice (up to 80 days). Most relevant to this proposal, we have obtained preliminary data showing that treatment of allogeneic islets in vitro (pre-injection) with these GW4869 suppressed donor MHC cross-dressing and nearly abrogated activation of alloreactive T cells in mice recipient of these islets. Contrasting with their role in rejection, exosomes have also been shown to promote immune tolerance. For instance, exosomes derived from FoxP3+ regulatory T cells (Treg-exosomes) suppress inflammation and can mediate immune tolerance of auto- and allo-antigens in rodents. On the other hand, to our knowledge, the tolerogenicity of exosomes produced by regulatory B cells (Breg-exosomes) has never been investigated. Our objectives are: 1) to investigate the nature of the cells and antigens involved in donor exosome release and cross-dressing after islet transplantation and, 2) test whether long-term islet allograft survival could be achieved through inhibition of donor exosome production and/or MHC cross-dressing or via recipient administration with tolerogenic exosomes. Aim 1. Investigate the mechanisms involved in donor antigen cross-dressing of recipient cells after islet transplantation Aim 2. Inhibit donor exosome release and cross-dressing in islet-transplanted mice Aim 3. Achieve long-term islet allograft survival using exosomes derived from regulatory cells We anticipate that our proposal will 1) bring new insights into the mechanisms underlying the initiation of alloimmunity and rejection process after pancreatic islet transplantation and, 2) set the path for the design of novel exosome-based tolerance protocols in islet transplantation and potentially autoimmune and other inflammatory immunological disorders.